光电关联显微镜技术及其在植物学研究中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0167

摘要/Abstract

摘要：

光电关联显微镜技术（correlative light and electron microscopy,CLEM）将光学显微镜的高灵敏度和大视场与电子显微镜的高分辨率相结合,弥补了各自成像的局限,可在原位获得更全面、更精细的定位及结构信息,近年来受到广泛关注。目前,该技术广泛应用于亚细胞结构与特定结构的观察、蛋白质的精确定位、囊泡运输及植物免疫等多个重要研究领域。本文总结了CLEM的概念和原理,对标记探针进行了总结分类,着重就该技术在植物学领域的应用以及存在的问题进行了讨论,并对该技术未来的发展与应用作出了展望。

关键词: 光电联用探针, 结构观察, 蛋白定位, 囊泡运输, 植物免疫

Abstract:

Correlative light and electron microscopy（CLEM）combines the high sensitivity and wide view of light microscopy with the excellent spatial resolution of the electron microscopy,and it overcomes the limitations of their respective imaging,enabling to obtain more comprehensive and fine positioning and structural information in situ,thus widespread attention has been paid to it in recent years. As a powerful attractive technique,CLEM has been widely used in the observation of subcellular structures and specific structures,precise localization of proteins,and the study of vesicle transport and plant immunity. Here,we summarize the concept and principle of CLEM and sum up the probes used in CLEM. Then we discuss the application of CLEM in the field of botany and point out the existing issues. Finally,we propose some aspects for the future development and application of CLEM.

Key words: CLEM probe, structural observation, protein localization, vesicle transport, plant immunity

武欣媛, 王广超, 林金星, 荆艳萍. 光电关联显微镜技术及其在植物学研究中的应用[J]. 生物技术通报, 2022, 38(1): 278-288.

WU Xin-yuan, WANG Guang-chao, LIN Jin-xing, JING Yan-ping. Correlative Light and Electron Microscopy and Its Application in Botanical Research[J]. Biotechnology Bulletin, 2022, 38(1): 278-288.

图/表 1

参考文献 70

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种类 Category	名称 Name	特点 Features	参考文献 Reference
真空中稳定的荧光探针 Stable fluorescent probes under vacuum	荧光染料：DAPI、Alex等 Fluorochromes：DAPI,Alexa,etc.	电子对比度低,电镜下观察不到	[19-20]
无机双对比探针 Inorganic dual-contrast probes	荧光纳米金 Fluoronanogold	渗透性好,在光镜和电镜下均可直接成像	[21-23]
无机双对比探针 Inorganic dual-contrast probes	量子点 Quantum dots	具有高电子密度、良好的光稳定性与抗漂白性,可同时进行不同组分的标记,在光镜和电镜中均可直接成像	[23-24]
基因编码的探针 Genetically encoded probes	双砷染料-四半胱氨酸系统 Tetracysteinebiarsenical	目的蛋白连接四半胱氨酸序列,双砷荧光团与之结合而具有强荧光,同时其对DAB具有光转化作用,也可实现在电镜下成像	[25-29]
	小型单线态氧气发生器 Mini singlet oxygen generator	分子量比GFP小,在蓝光激发下能够发出绿色荧光,通过氧化DAB形成电镜下可见的嗜锇小体	[30]
	增强型抗坏血酸过氧化酶 Enhanced ascorbate peroxidase	当APEX与荧光蛋白结合时,可在活细胞中表达出荧光信号,经过化学固定以及添加DAB和H₂O₂,可以产生EM可见的反应产物	[31-32]
	绿色荧光蛋白及其衍生物 Green fluorescent protein and its derivatives	需要通过DAB进行光电转化;也可通过高压冷冻与GMA包埋或者Tokuyasu保护荧光蛋白的荧光,实现光电关联	[33-35]
	基因编码的化学诱导探针 Genetically-encoded chemically-inducible probes	荧光重组电子致密的铁蛋白颗粒与感兴趣的蛋白连接,雷帕霉素诱导表达,直接在光镜与电镜下成像	[36]
	光转换荧光蛋白mEos Photoconvertible fluorescent protein mEos	电镜制样后,超薄切片中仍保持荧光活性	[37-38]

种类 Category	名称 Name	特点 Features	参考文献 Reference
真空中稳定的荧光探针 Stable fluorescent probes under vacuum	荧光染料：DAPI、Alex等 Fluorochromes：DAPI,Alexa,etc.	电子对比度低,电镜下观察不到	[19-20]
无机双对比探针 Inorganic dual-contrast probes	荧光纳米金 Fluoronanogold	渗透性好,在光镜和电镜下均可直接成像	[21-23]
无机双对比探针 Inorganic dual-contrast probes	量子点 Quantum dots	具有高电子密度、良好的光稳定性与抗漂白性,可同时进行不同组分的标记,在光镜和电镜中均可直接成像	[23-24]
基因编码的探针 Genetically encoded probes	双砷染料-四半胱氨酸系统 Tetracysteinebiarsenical	目的蛋白连接四半胱氨酸序列,双砷荧光团与之结合而具有强荧光,同时其对DAB具有光转化作用,也可实现在电镜下成像	[25-29]
	小型单线态氧气发生器 Mini singlet oxygen generator	分子量比GFP小,在蓝光激发下能够发出绿色荧光,通过氧化DAB形成电镜下可见的嗜锇小体	[30]
	增强型抗坏血酸过氧化酶 Enhanced ascorbate peroxidase	当APEX与荧光蛋白结合时,可在活细胞中表达出荧光信号,经过化学固定以及添加DAB和H₂O₂,可以产生EM可见的反应产物	[31-32]
	绿色荧光蛋白及其衍生物 Green fluorescent protein and its derivatives	需要通过DAB进行光电转化;也可通过高压冷冻与GMA包埋或者Tokuyasu保护荧光蛋白的荧光,实现光电关联	[33-35]
	基因编码的化学诱导探针 Genetically-encoded chemically-inducible probes	荧光重组电子致密的铁蛋白颗粒与感兴趣的蛋白连接,雷帕霉素诱导表达,直接在光镜与电镜下成像	[36]
	光转换荧光蛋白mEos Photoconvertible fluorescent protein mEos	电镜制样后,超薄切片中仍保持荧光活性	[37-38]